lishufeng-高級(jí)生化實(shí)驗(yàn)ppt課件

1、高級(jí)生化實(shí)驗(yàn)實(shí)驗(yàn)部分1.實(shí)驗(yàn)17：脲酶的分別純化及比活性測(cè)定2.實(shí)驗(yàn)19：重組pGEX-X載體的構(gòu)建及表達(dá)實(shí)際部分第2章 電泳技術(shù)影響電泳的要素和PAGE，SDS-PAGE的原理第3章 層析技術(shù)層析的概念及種類離子交換層析、凝膠層析和親和層析的原理第4章 蛋白質(zhì)的分別純化第7章 DNA的制備與分析質(zhì)粒DNA的制備和DNA的瓊脂糖凝膠電泳第8章 聚合酶鏈反響第1、2節(jié)-根本原理及反響體系第10章 基因工程重點(diǎn)李淑鋒，Ph.D, associate professor: shufengli3000yahoo留意上課時(shí)間，保證出勤率實(shí)驗(yàn)室儀器運(yùn)用規(guī)范移液器、離心機(jī)等，損壞賠償實(shí)驗(yàn)：分組、試劑、實(shí)驗(yàn)講

2、義實(shí)驗(yàn)平安，儀器、試劑、菌液5. 考試：實(shí)驗(yàn)40操作報(bào)告 閉卷606. 值日DAY 1:配試劑配試劑DNA的提取及鑒定的提取及鑒定高壓，滅菌培育皿、槍頭、離心管、試劑高壓，滅菌培育皿、槍頭、離心管、試劑倒平皿加抗生素倒平皿加抗生素Kan,不加抗生素不加抗生素 每排每排4LK, 2LB 50mg/ml kana加到加到LB中中kana 1:1000稀釋稀釋銜接：質(zhì)粒片斷目的基因銜接：質(zhì)粒片斷目的基因 每排每排2個(gè)銜接反響個(gè)銜接反響ddH2O 12ul ddH2O 12ul X-DNA(X-DNA(目的基因目的基因 3ul 3ul pET-28apET-28a質(zhì)粒片斷質(zhì)粒片斷 2ul 2ul 10

3、10buffer 2ul buffer 2ul T4 ligase 1ulT4 ligase 1ul14-160C14-160C過夜過夜20ul20ul 每組接種每組接種DH5a 1支支 接種接種BL21 1支支 10ul菌液菌液+2ml LB, 第二天第二天7：30轉(zhuǎn)接轉(zhuǎn)接 1ml+20mlLB分別預(yù)備制備感受態(tài)，兩個(gè)銜接反響用來轉(zhuǎn)化分別預(yù)備制備感受態(tài)，兩個(gè)銜接反響用來轉(zhuǎn)化實(shí)驗(yàn)內(nèi)容1.重組載體的構(gòu)建及表達(dá)6天2.脲酶的分別純化及活性測(cè)定2天總體實(shí)驗(yàn)安排上午下午第一天配試劑挑DH5單克隆，連接反應(yīng)第二天感受態(tài)細(xì)胞的制備轉(zhuǎn)化第三天觀察結(jié)果講質(zhì)粒的提取鑒定原理挑單克隆過夜培養(yǎng)第四天質(zhì)粒的提取、酶切

4、瓊脂糖凝膠電泳挑鑒定正確的克隆過夜培養(yǎng)（3ml)第五天擴(kuò)大培養(yǎng)（30ml)IPTG誘導(dǎo)，0，1，2，3小時(shí)取樣SDS-PAGE第六天蛋白純化試劑配制裝柱、脲酶粗提液制備第七天層析蛋白濃度及活性測(cè)定第八天電泳檢測(cè)、打掃實(shí)驗(yàn)室DH5aDH5a感受態(tài)細(xì)胞的制備：感受態(tài)細(xì)胞的制備：P159P159 轉(zhuǎn)種轉(zhuǎn)種1 1：4040 繼續(xù)振蕩培育繼續(xù)振蕩培育2.5h 2.5h 制備感受態(tài)細(xì)胞制備感受態(tài)細(xì)胞CaCl2)CaCl2)銜接反響終止銜接反響終止轉(zhuǎn)化，涂板，轉(zhuǎn)化，涂板，3737度過夜培育度過夜培育 DH5DH5：感受態(tài)一塊：感受態(tài)一塊LB,LB,感受態(tài)一塊感受態(tài)一塊LK LK 轉(zhuǎn)化菌轉(zhuǎn)化菌一塊一塊LK,

5、BL21LK, BL21同樣同樣DAY 2:LBLB平皿平皿DH5aDH5a感受態(tài)細(xì)胞感受態(tài)細(xì)胞25UL25ULLKLK平皿平皿DH5aDH5a感受態(tài)細(xì)胞感受態(tài)細(xì)胞25UL25ULLKLK平皿平皿轉(zhuǎn)化菌液轉(zhuǎn)化菌液50-100UL50-100ULDAY 3:看結(jié)果，配試劑誘導(dǎo)表達(dá)及鑒定用看結(jié)果，配試劑誘導(dǎo)表達(dá)及鑒定用 接種：質(zhì)粒提取用接種：質(zhì)粒提取用2人人/管管LBLB平皿平皿DH5aDH5a感受態(tài)細(xì)胞感受態(tài)細(xì)胞25UL25ULLKLK平皿平皿DH5aDH5a感受態(tài)細(xì)胞感受態(tài)細(xì)胞25UL25ULLKLK平皿平皿轉(zhuǎn)化菌液轉(zhuǎn)化菌液50-100UL50-100UL. . . . . . . . . .

6、？DAY 4:質(zhì)粒提取質(zhì)粒提取P160P160：堿裂解法：堿裂解法質(zhì)粒的酶切質(zhì)粒的酶切: 20ul, 370C,1H: 20ul, 370C,1H 瓊脂糖電泳檢測(cè)瓊脂糖電泳檢測(cè)P161 ： 接種：誘導(dǎo)表達(dá)用每一組接種：誘導(dǎo)表達(dá)用每一組/管，需轉(zhuǎn)種管，需轉(zhuǎn)種單酶切：?jiǎn)蚊盖校褐亟M質(zhì)粒重組質(zhì)粒DNA 5ulDNA 5ulBamH 1 1ulBamH 1 1ul1010buffer 2ulbuffer 2ulddH2O 12ulddH2O 12ul雙酶切：雙酶切：重組質(zhì)粒重組質(zhì)粒DNA 5ulDNA 5ulBamH 1 1ulBamH 1 1ulXhol 1 1ulXhol 1 1ul1010buff

7、er 2ulbuffer 2ulddH2O 11ulddH2O 11ul 轉(zhuǎn)種轉(zhuǎn)種1 1：4040 培育培育3h3h，參與，參與IPTGIPTG誘導(dǎo)誘導(dǎo) 不同時(shí)間取菌不同時(shí)間取菌0h1h0h1h、2h2h、3h3h取菌取菌 電泳電泳DAY 5， DAY 6：制備制備PAGEPAGE凝膠凝膠P163P163：下層膠、上層膠：下層膠、上層膠誘導(dǎo)表達(dá)及誘導(dǎo)表達(dá)及PAGEPAGE電泳：電泳： Question：構(gòu)建的載體為何需求進(jìn)展酶切及瓊脂糖電泳電泳的構(gòu)建的載體為何需求進(jìn)展酶切及瓊脂糖電泳電泳的鑒定？鑒定？除了以上的鑒定方法，還能知道哪些其他手段鑒定除了以上的鑒定方法，還能知道哪些其他手段鑒定重組載

8、體？重組載體？除了選擇除了選擇BamH 1 BamH 1 、 Xhol 1 Xhol 1 ，還可以選擇哪些酶進(jìn)，還可以選擇哪些酶進(jìn)展鑒定？展鑒定？如何分析瓊脂糖電泳的結(jié)果？如何分析瓊脂糖電泳的結(jié)果？參與參與IPTGIPTG誘導(dǎo)蛋白表達(dá)的原理？誘導(dǎo)蛋白表達(dá)的原理？SDS-PAGESDS-PAGE電泳分別蛋白質(zhì)的機(jī)理？電泳分別蛋白質(zhì)的機(jī)理？Discovery of the Double Helix 1953, James Watson and Francis Crick are credited with building the first model of DNA. They won the

9、Nobel Prize in 1962The Central Dogma常用分子生物學(xué)技術(shù)常用分子生物學(xué)技術(shù) 基因克隆基因克隆 DNADNA序列測(cè)定序列測(cè)定 核酸分子雜交核酸分子雜交 PCRPCR 轉(zhuǎn)基因生物轉(zhuǎn)基因生物 DNADNA芯片芯片Why gene Cloning?Isolation and manipulation of fragments of an organisms genomeMolecular analysis of proteins or other interested gene products Impossible by direct purification DN

10、A cloning Impossible by direct isolationMake all possible ! Make all possible ! Basic procedureof DNA cloningVector基因克隆基因克隆(gene cloning):是指采用人工方是指采用人工方法將目的基因與載體法將目的基因與載體DNA在體外進(jìn)展重在體外進(jìn)展重組，并將重組后的組，并將重組后的DNA引入宿主細(xì)胞中引入宿主細(xì)胞中進(jìn)展擴(kuò)增或表達(dá)的過程。進(jìn)展擴(kuò)增或表達(dá)的過程。 又稱重組又稱重組DNADNA技術(shù)技術(shù)DNA recombination DNA recombination techn

11、ique) technique) 或或基因工程基因工程(genetic engineering(genetic engineering基因工程的操作過程基因工程的操作過程目的基因的分別；目的基因的分別；載體的選擇和銜接；載體的選擇和銜接；重組重組DNADNA導(dǎo)入受體細(xì)胞；導(dǎo)入受體細(xì)胞；重組重組DNADNA的挑選和鑒定；的挑選和鑒定；克隆基因的表達(dá)?？寺』虻谋磉_(dá)。One component of the bacterial restriction-modification system.Restriction endonuclease: 1) recognize short, symmetri

12、cal DNA sequence; 2) cut DNA backbone at a specific site within that sequence (kill foreign DNA).Mythylase: methylates C or A of the cellular DNA Section 1: Tool enzymes:1.Restriction endonucleaseRecognition sequencesRestriction enzymes1)Recognize 4-8 bp palindromic sequence. 2)Highly specific.Comme

13、rcially availableRequire Mg2+ for enzymatic activityRestriction sequences5 protruding ends3 protruding ends5-CCCGGG-33-GGGCCC-55-CCC-OH3-GGG- pp -GGG-3OH-CCC-5+SmaIblunting ends/粘性末端Cohensive ends/平末端Isoschizomers/同功異源酶:來源不同，但能識(shí)別和切割同一位點(diǎn)，這些酶稱。：Isoaudamers/同尾酶：識(shí)別序列不同，但能產(chǎn)生一樣的粘性末端的酶自我復(fù)制才干；自我復(fù)制才干；易于從宿主細(xì)胞

14、分別；易于從宿主細(xì)胞分別；多克隆位點(diǎn)；多克隆位點(diǎn)；選擇標(biāo)志。選擇標(biāo)志。載體條件：載體條件：1 Plasmid vectors2 Bacteriophage vectors3 Virus4 Cosmids and YACs Cloning vectors:Multiple restriction sites enable the convenient insertion of target DNA into a vector Two examples: phage bacteriophage replacement vector M13 phageM13 phage vectorCloning

15、in M13Hybrid plasmid-M13 vectorsDNAProtein coatcoscosNonessential regionLong (left)armshort (right)armExogenous DNA(20-23 kb)12bp Viruses that can infect bacteria. 48.5 kb in lengthLinear or circular genome (cos ends)Lytic phase (Replicate and release)Lysogenic phase (integrate into host genome)5-CG

16、GGGCGGCGACCTCG-33-GCCCCGCCGCTGGAGC-5 Cleavage Ligation(during packaging) (after infection) GGGCGGGCGACCTCG-35-CG + GC-53-GCCCCGCCGCTGGAThe phage cos endsCircular form Linear form Cloning large DNA fragments 20 kb) Cosmid vectors 粘粒載體粘粒載體 YAC 酵母人工染色體酵母人工染色體vectors Selection in S. cerevisiae (啤酒酵母啤酒酵母

17、Yeast Artificial ChromosomeDigestionLigationPackaging and infectFormation of a cosmid cloneEssential components of YAC vectors :Centromere (CEN著絲粒著絲粒),telomere (TEL端粒端粒) autonomous replicating sequence (ARS) ampr and markers such as TRP1 and URA3. Recognition sites of restriction enzymesCan accommod

18、ate genomic DNA fragments of more than 1 MbYeast selection載體種類：載體種類： Plasmid Phage and Virus Cosmid YAC and BAC1.acquiring target gene 1) screening from genomic library;2) screening from cDNA library;3) by PCR4) Chemical synthesis;2 . v e c t o r s e l e c t i o n3. ligation 1) cohesive end ligation

19、2) blunt end ligation3) through linker(人工接頭人工接頭4) adding homopolymeric tail同聚物加尾同聚物加尾.DNA ligation strategy4. Introduce recombinant 4. Introduce recombinant DNA to host cellsDNA to host cellsCompetent cells: E. coli cells treated with Ca2+ solution are susceptible to take up exogenous DNA. Enzymes i

20、nvolved in host cell defending, such as restriction-modification system are suppressed. Transformation: a process of uptake of exogenous DNA by competent cells. Heat-shock: After the DNA is uptaken, the cells shall be put at 42oC for 1 min in order to induce the suppressed enzymes for cell defending

21、重組重組DNADNA導(dǎo)入受體菌導(dǎo)入受體菌 1.1.轉(zhuǎn)化轉(zhuǎn)化transformationtransformation：將質(zhì)粒：將質(zhì)粒DNADNA導(dǎo)入原核細(xì)胞的過程化學(xué)法及電轉(zhuǎn)化導(dǎo)入原核細(xì)胞的過程化學(xué)法及電轉(zhuǎn)化等等2.2.感染感染infection):infection):經(jīng)過噬菌體將外源經(jīng)過噬菌體將外源DNADNA導(dǎo)入原核細(xì)胞的過程；導(dǎo)入原核細(xì)胞的過程；3.3.轉(zhuǎn)染轉(zhuǎn)染(transfection)(transfection)：將外源：將外源DNADNA導(dǎo)入導(dǎo)入真核細(xì)胞的過程。真核細(xì)胞的過程。5.selection of recombinant DNA 1. antibiotics resista

22、nce抗性抗性 2.insertion inactivation插入失活插入失活3. a-complementation a -互補(bǔ)互補(bǔ)4.immunology methods免疫學(xué)方法免疫學(xué)方法5.southern bloting6.PCR7.restriction digestion(酶切鑒定酶切鑒定)8.Sequencing(測(cè)序測(cè)序)基因工程的操作過程基因工程的操作過程目的基因的分別；目的基因的分別；載體的選擇和銜接；載體的選擇和銜接；重組重組DNADNA導(dǎo)入受體細(xì)胞；導(dǎo)入受體細(xì)胞；重組重組DNADNA的挑選和鑒定；的挑選和鑒定；克隆基因的表達(dá)。克隆基因的表達(dá)。pET28a銜接反響 在

23、1.5ml離心管中參與 ddH2O12ul T4 DNA ligase1ul X-DNA3ul pGEX(vector)2ul 10buffer2ul 混勻輕彈或反復(fù)用槍吹吸，16C過夜感受態(tài)的制備1.5菌液沉淀，加0.5ml CaCl2 混勻離心，4000rpm, 5分鐘離心，4000rpm, 5分鐘沉淀，加0.5ml CaCl2 混勻冰浴，30分鐘離心，4000rpm, 5分鐘沉淀，加100ul CaCl2 混勻立刻便用4度短期保管-70度長(zhǎng)期保管加15%-30%甘油轉(zhuǎn)化10ul銜接液+50-100ul感受態(tài)42度水浴熱激90秒混勻，冰浴，30分鐘加0.5-1ml LB，混勻37度搖床，培

24、育1小時(shí)倒上清，重懸沉淀，涂平板5000rpm, 離心3分鐘10ul剩余LBLB+Kan50ul感受態(tài)LB+Kan5.selection of recombinant DNA 1. antibiotics resistance抗性抗性 2.insertion inactivation插入失活插入失活3. a-complementation a -互補(bǔ)互補(bǔ)4.immunology methods免疫學(xué)方法免疫學(xué)方法5.southern bloting6.PCR7.restriction digestion(酶切鑒定酶切鑒定)8.Sequencing(測(cè)序測(cè)序)抗藥性挑選重組體抗藥性挑選重組體Am

25、picillin resistant? yes yesTetracycline resistant? No yesB X BBBXAmproriAmprTcrori2.insertional inactivationAmprTcroripBR322BReplica plating: transfer of the colonies from one plate to another using absorbent pad or velvet (絨布絨布).transfer of colonies+ampicillin+ ampicillin+ tetracyclineThese colonie

26、s have bacteria with recombinant plasmidAmproripUC18(3 kb)MCS (Multiple cloning sites,多克隆位點(diǎn)多克隆位點(diǎn)Lac promoterlacZScreening by insertional inactivation of the lacZ geneThe insertion of the target gene interrupts the ORF of lacZ gene. .a-complementation.a-complementationl Plasmids encodes lacZ(N-termin

27、al a-peptide of galactosidase). l Host strain encoding only the C-terminal portion of -galactosidase.l N-terminal + C-terminal = active -galactosidase.IPTGX-gal(substrate of the enzyme)lac promoterBlue producta-complementationa-complementationRecreated vector: blue transformantsRecombinant plasmid c

28、ontaining inserted DNA: white transformantsRecreated vector (no insert)Recombinant plasmid (contain insert).Identify the protein product of an interested gene1) Protein activity2) Western blotting.用核酸雜交法進(jìn)展分析鑒定：用核酸雜交法進(jìn)展分析鑒定：6.Restriction mapping: digestion with restriction enzymes.7.Sequencing the cl

29、oned DNA8. PCR analysis質(zhì)粒 M 雙 雙 單 單15,00010,0007,5005,0002,5001,000250質(zhì)粒提取-堿裂解法1.5菌液沉淀，加100ul溶液I離心，6000rpm, 3分鐘棄上清充分充分混勻，冰上5分鐘加200ul溶液II顛倒混勻輕柔冰上5分鐘加150ul溶液III顛倒混勻，冰上5分鐘離心，12000rpm, 10分鐘用槍吸400ul上清至新管加1ml無水乙醇，混勻，冰上5-10分鐘沉淀，參與1ml 70%酒精離心，12000rpm, 10分鐘離心，1200rpm, 5分鐘用槍汲取剩余液體棄上清1200rpm瞬時(shí)離心沉淀，加20ulTE+1ul

30、RNAase開蓋，晾5-10分鐘質(zhì)粒酶切反響 在1.5ml離心管中參與單酶切(ul) 雙酶切(ul)ddH2O141310buffer2ul2質(zhì)粒3ul3BamH I11Xho I/1Total 2020充分混勻后，瞬時(shí)離心，37度保溫1-2小時(shí)瓊脂糖凝膠電泳l 溶膠用TAE緩沖液，充分融化l 稍冷卻后倒膠l 加樣樣品要加樣品緩沖液至1l 電泳1-10V/CM)l 染色與察看拍照質(zhì)粒 M 雙 雙 單 單15,00010,0007,5005,0002,5001,000250Lane 2: Multimeric forms of supercoiled plasmid DNA (pTZ19) wh

31、ich may be observed with some host strains, and should not be mistaken for genomic DNA. Lane 3: Linearized form of plasmid pTZ19 after restriction digestion with EcoRI.Lane 4: Sample contaminated with bacterial chromosomal DNA, Genomic DNA contamination can easily be identified by digestion of the s

32、ample with EcoRI. A smear is observed, in contrast to the linear band seen after digestion of multimeric plasmid forms.Lane 5: EcoRI digestion of a sample contaminated with bacterial genomic DNA which gives a smear above the plasmid DNA.Lane 1: Supercoiled (lower band) and open circular form (upper

33、band) of the high-copy plasmid pUC18 with an additional band of denatured supercoiled DNA migrating just below the supercoiled form. This form may result from prolonged alkaline lysis with Buffer P2 and is resistant to restriction digestion蛋白誘導(dǎo)表達(dá)取樣1.5ml至1.5ml離心管2ml過夜培育菌液37度震蕩培育3小時(shí)轉(zhuǎn)移1ml至30ml培育基/Kan加I

34、PTG至0.1-1mM37度震蕩培育1小時(shí)取樣0.8ml至1.5ml離心管37度震蕩培育2小時(shí)取樣0.5ml至1.5ml離心管SDS-PAGE樣品制備棄上清再 1200rpm瞬時(shí)離心離心，4000rpm, 5分鐘混勻后至沸水浴中煮3-5分鐘用槍吸去上清，加50ulH2O重懸沉淀4度或-20度保管菌液參與50ul 2SDS樣品緩沖液SDS-PAGE過程l配膠l樣品制備l加樣l電泳200Vl染色：考馬斯亮藍(lán)染色或銀染dH2O：3.3ml30%丙烯酰胺：4.0ml1.0M Tris-Cl(pH8.8)：2.5m110SDS：0.1 ml10%過硫酸銨：0.1 mlTEMED：0.005 mldH2O

35、: 3.4ml 30%丙烯酰胺：0.85ml1M Tris-CI(pH68):0.625m110SDS：0.05ml10%過硫酸銨：0.05ml TEMED：0.005ml制備濃縮膠(5%) 制備分別膠(12) 配膠CH2CH CNH2OCH2CH CNH2OCH2CH CNH2OCH2+聚協(xié)作用催化劑CH2CHCNHCHx CH2 CH2OCOCH2NHCH2CHCHx CH2 CH2CNH2OCNH2O丙烯酰胺N,N-甲叉雙丙烯酰胺聚丙烯酰胺凝膠聚丙烯酰胺凝膠電泳polyacrylamide gel electrophoresis，PAGE l 單體:丙烯酰胺Acr+ 甲叉雙丙烯酰胺Bis

36、 l 催化劑：過硫酸銨+四甲基乙二胺TEMED凝膠的聚合SDS-PAGE分離膠濃度最佳分離范圍6%膠50-150kD8%膠30-90kD10%膠20-80kD12%膠12-60kD15%膠10-40kD膠濃度與有效分別范圍PAGE分別效應(yīng)l濃縮效應(yīng) l電荷效應(yīng) l分子篩效應(yīng) 故具有高分辨率 Samples must be previously boiled 5 minutes in sample buffer containing: SDS (CH3-(CH2)10-CH2OSO3- Na+), 1 molecule binds every 2 amino acids residues b-M

37、ercaptoethanol Sucrose or Glycerol Ionizable tracking dye (i.e., bromophenol blue) Separate protein according to sizeSDS-PAGE 蛋白質(zhì)的變性SDS-PAGE過程l配膠l樣品制備l加樣l電泳200Vl染色：考馬斯亮藍(lán)染色或銀染實(shí)驗(yàn)二蛋白質(zhì)分別純化脲酶的分別純化及比活性測(cè)定l制備脲酶粗提液l裝柱l凝膠過濾層析l蛋白質(zhì)含量檢測(cè)l蛋白質(zhì)活性檢測(cè)脲酶活性測(cè)定原理CO(NH2)2 + H2O2NH3 + CO2脲酶NH4OH + 2(HgI22KI) + 3NaOHO NH2I +

38、4KI + 3NaI + 3H2OHgHg硫代雙汞氨黃色奈氏試劑反響1反響2脲酶活性高生成氨就多黃色亦深制備脲酶粗提液稱0.5克黃豆粉于小三角瓶中加2.5ml32%丙酮，震蕩10min離心3000轉(zhuǎn)，10分鐘上清液轉(zhuǎn)至新離心管，加4倍體積冷丙酮離心3000轉(zhuǎn)，5分鐘沉淀，待丙酮揮發(fā)后，加1.2ml蒸餾水溶解離心3000轉(zhuǎn)，10分鐘上清/粗提液凝膠過濾層析l 裝柱：堅(jiān)持豎直，防分層、干裂l 平衡：裝好后用蒸餾程度衡l 加樣：加0.6ml粗提液l 洗脫與搜集：用蒸餾水洗脫，3ml/15分鐘/管，搜集12管l 蛋白質(zhì)含量檢測(cè)：A280測(cè)定l 粗提液稀釋20倍再測(cè)，測(cè)完A280，務(wù)必保管樣品l 蛋白濃

39、度mg/ml=A2800.75l 酶活性檢測(cè)蛋白含量測(cè)定空白洗脫液粗提液(1:20稀釋)1239101112A280蛋白(mg/ml)1.硫酸銨規(guī)范曲線制造管號(hào) 試劑12345670.001M硫酸銨(ml)-含NH3的mol數(shù)0.811.2-H2O（ml)3混勻奈氏試劑0.75ml, 混勻，測(cè)A480A480脲酶活性檢測(cè)-1硫酸銨規(guī)范曲線制造含NH3的mol數(shù)1.2 1.0 0.8 0.6 0.4 0.2A4800 0.2 0.4 0.6 0.8 1.0 1.2NH3的mol數(shù) = A480 K脲酶活性檢

40、測(cè)-2空白洗脫液粗提液(1:20稀釋)12391011123%尿素(ml)0.50.1M PBS1.01.01.01.01.01.01.01.01.01.037度保溫15分鐘酶液-H2O0.5-混勻，37度保溫15分鐘向各管加入1M HCl 0.5ml,放置5分鐘，加1M NaOH 0.5ml終止反應(yīng)2. 酶促反響脲酶活性檢測(cè)-3空白洗脫液粗提液(1:20稀釋)1239101112上述反應(yīng)液0.1H2O2.52.52

41、.2.9奈氏試劑每管加0.75ml,混勻A4803. 顯色與測(cè)定酶活性計(jì)算空白洗脫液粗提液(1:20稀釋)1239101112A480NH3(umol)活性/ml洗脫液活性/管蛋白(mg/ml)比活性單位：NH3(umol)數(shù)/mL洗脫液h顯色用體積 活性/ml洗脫液 = NH3(umol)數(shù) 3.0取酶促反響液ml數(shù)10.56015規(guī)定1小時(shí)測(cè)15分鐘測(cè)0.5ml 規(guī)定1ml酶反響液3ml 活性/管 =活性/ml洗脫液 3比活性 =每毫升洗脫液的活性每毫升洗脫液的蛋白含量General principles of protein purificationlComplica

42、ted and specificl“Black artSELECT SOURCE OF MATERIAL Concentration: choose tissue, organism with high production/concentration of target protein Developmental stage: Does level of protein change with development? Subcellular localization Use of expression systemProtein purification Pretreatment/預(yù)處置

43、Rough fractionation/粗分級(jí) Fine fractionation/細(xì)分級(jí)電泳電泳 前前處處置置粗粗分分別別細(xì)細(xì)分分別別生物組織生物組織 無細(xì)胞提取液無細(xì)胞提取液破碎、差速離心、溶解膜蛋白等破碎、差速離心、溶解膜蛋白等選擇性沉淀法選擇性沉淀法親和親和層析層析離子交離子交換層析換層析精制后的蛋白質(zhì)精制后的蛋白質(zhì)可結(jié)晶純化可結(jié)晶純化凝膠凝膠過濾過濾疏水疏水層析層析吸附吸附層析層析分段鹽析、有機(jī)溶劑分段鹽析、有機(jī)溶劑分級(jí)沉淀、凝膠層析分級(jí)沉淀、凝膠層析Methods for Protein PurificationTake advantage of the following pr

44、operties of different protein.molecular weight/MWsolubilitycharge difference ligand specificity親和層析 selective adsorptionhydrophobic propertydialysis/透析& ultrafiltration/超濾法：Remove small molecules or ions density gradient centrifugation密度梯度離心gel filtration/凝膠過濾法1. According to size and shape semi-per

45、meable membrane. Pores of membrane are of a certain size. Protein stays in; small molecules pass through.Dialysis透析Gel Filtration凝膠過濾層析l Column made of porus beadsl Separates in terms of sizel Big proteins elute first2. In terms of solubilityisoelectric precipitationSalting in and salting outOrganic

46、 solvent precipitationSalting in / Salting outl Salting INl low concentrations of salt usually increases the solubility of charged macromolecules.l Salting OUTl high concentrations of added salt lowers the solubility of and they come out of solution.Effect of K2SO4 on the solubility of Hb-CO離子強(qiáng)度溶解度1) electr